We thank the NCFPB Integrated Core Facility for Functional Genomics (Taipei, Taiwan) and the NRPB Pharmacogenomics Lab (Taipei, Taiwan) for their technical support. This work was supported by the Ministry of Science and Technology, Taiwan, R.O.C. [grant number MOST109-2314-B-002 -186 to K.-Y.S., MOST 107-2320-B-002-016-MY3 to S.-Y.C., MOST 110-2320-B-002-043 to W.-h.F.]. H.-L. C. is a recipient of a doctoral fellowship from National Taiwan University. Funding for open access charge: Ministry of Science and Technology, R.O.C.

1. Substrate/template preparation
<ol>
	<li>Design uracil substrate/template duplex with a balanced G+C content of ~50 &#177; 10% and minimum melting temperature of 50 &#176;C for the duplex region. 
	NOTE: One nucleotide difference between 18 nt substrates and 19 nt templates (Table 1 and Figure 1) helps better MS signal interpretation and appropriate annealing. The template strand serves as complementary DNA to generate A-U or G-U mismatches (Table 1) but can also be used as a reference signal in MS measurements. The use of HPLC-purified synthetic oligonucleotides is satisfactory for this study.</li>
	<li>Dissolve DNA in 1 mM EDTA and 10 mM Tris-HCl(pH 8.0 at 25 &#176;C) (TE) at a concentration of 100 &#181;mol/L as a stock and store at -20 &#176;C. Dilute this 20 &#181;L stock with TE to a final volume of 800 &#181;L (25-fold dilution to 4 &#181;mol/L). Measure the absorbance of the DNA solution in a UV-visible spectrophotometer at &#955; = 260 nm to ensure the manufacturer&#39;s assigned concentration. For example: A260 = 0.204 for 4 &#181;mol/L of U+9 and A260 = 0.192 for 4 &#181;mol/L of T1 (Table 1).</li>
	<li>Perform MALDI-TOF MS analysis (sections 4-6) for oligonucleotide quality control by inspecting unique peak signals at designated m/z values as well as by checking that the signal-to-noise ratio is &#62;100 (Figure 1B and Figure 1D).</li>
</ol>
2. DNA glycosylase assay
<ol>
	<li>Using a G:U substrate of T1/U+9 duplex (Table 1 and Figure 1A) for the glycosylase reaction, for example, in a 1.5 mL sterile microcentrifuge tube, add 70 &#181;L of H2O, 10 &#181;L of 10x UDG reaction buffer, 5 &#181;L of T1 stock, and 5 &#181;L of U+9 stock (step 1.2.). 
	NOTE: Choose the correct type of micropipette and follow the manufacturers&#39; instructions to handle the required volume. For example, use a 2 &#181;L pipette to dispense 0.1-2 &#181;L of liquid, use a 10 &#181;L pipette to dispense 2-10 &#181;L of liquid, and use a 100 &#181;L pipette to dispense 20-100 &#181;L of liquid to ensure accuracy and precision of the results. The described volume of the reagent mix is for 10 assays; adjust the volume for the desired number of reactions. The 1x UDG reaction buffer contains 1 mM EDTA, 1 mM dithiothreitol, and 20 mM Tris-HCl (pH 8.0 at 25 &#176;C). See the Table of Materials for the source of 10x UDG reaction buffer.</li>
	<li>Securely close the tube; incubate in a water bath for 30 min at 65 &#176;C, then for 30 min at 37 &#176;C, and finally on ice for 3 min to ensure proper annealing of the substrate/template duplex.</li>
	<li>In a 1.5 mL sterile microcentrifuge tube, add 49 &#181;L of ice-cold 1x UDG reaction buffer and 1 &#181;L of UDG (5,000 units/mL; see the Table of Materials), diluting to 0.1 units/&#181;L. Make serial dilutions with 1x UDG buffer to the desired enzyme concentrations of 0.05, 0.02, or 0.01 units/&#181;L. Always keep the diluted UDG on ice. 
	NOTE: In a 10 &#181;L reaction, 0.1 units of UDG can cleave more than 30 pmol of uracil from a T1/U+9 duplex in 3 min.</li>
	<li>In a 1.5 mL sterile microcentrifuge tube, add 9.0 &#181;L of substrate mix from step 2.2 and prewarm the tube to 37 &#176;C. Add 1.0 &#181;L of the diluted UDG from step 2.3. Use a timer to time the reaction and flick the tube to mix contents. 
	NOTE: For a unit definition assay, the incubation time is 30 min. For a kinetic assay, use a time-course analysis of 0.5, 1, 2, 3, 5 min to obtain the initial rate. For a UGI inhibition assay, time the reaction for 15 min.</li>
	<li>Centrifuge the tubes with the reaction mixture for 3-5 s at 3,200 &#215; g at ambient temperature. Then, transfer the reaction immediately to 37 &#176;C.</li>
	<li>Reaction termination
	<ol>
		<li>Prepare solutions of 0.25 M HCl and 0.23 M Tris base. In a 15 mL test tube, add 10 mL of a solution of 1 mM EDTA and 20 mM Tris-HCl (pH 8.0 at 25 &#176;C) to mimic 1x UDG reaction buffer. Acidify with 1 mL of 0.25 M HCl and check with a pH meter to ensure the pH is ~2 &#177; 0.5. Neutralize with 1 mL of 0.23 M Tris base and check with a pH meter for a final pH of 6.5 &#177; 0.5. 
		NOTE:&#160;Using pH test strips is a quick and easy way to reconfirm the pH levels of the reagent mix.</li>
		<li>Add 1 &#956;L of 0.25 M HCl to acidify the 10 &#956;L reaction mixture to inactivate the enzyme and place it on ice for 6 min. Add 1 &#181;L of 0.23 M Tris base to neutralize the DNA products to avoid AP site breakage by prolonged exposure to acid. Add 13 &#181;L TE to increase the volume of the product mixture for matrix chip transfer and then place it on ice. 
		NOTE: The AP product is chemically unstable and should be analyzed by MALDI-TOF MS within 2 days. After prolonged storage for more than a week, the accumulation of a substantial portion of strand breaks occurs due to &#946;/&#948; elimination reactions of the AP products (Figure 1D-G).</li>
	</ol>
	</li>
	<li>Transfer all the 25 &#181;L UDG reaction products from microcentrifuge tubes to a 384-well microtiter plate. 
	&#8203;NOTE: High concentrations of cations, such as sodium or potassium in buffers, generate interference in the MALDI-TOF MS analysis and thus require desalting. As E. coli UDG reaction buffer contains very low concentrations of cations, desalting is not necessary. However, modify this protocol to measure other DNA glycosylase reactions containing metal cations that require desalting as described previously35.</li>
</ol>
3. Transfer UDG reaction products to a matrix chip
<ol>
	<li>Open the door of the nanoliter dispenser (see the Table of Materials) and load the 384-well microtiter plate from step 2.7 onto the plate holder of the deck.</li>
	<li>Insert the matrix chip array into the corresponding scout plate position. Place the loaded scout plate on the processing deck of the nanoliter dispenser and close the door.</li>
	<li>Touch the run button on the transfer screen, and wait for the instrument to start dispensing samples from the 384-well microtiter plate to the matrix chip.</li>
	<li>Use the Vision tab option to show the image of the chip and the dispense volumes for each spot during dispensing. Ensure the spotted volume on the chip is in the range of 5-10 nL.</li>
</ol>
4. Setup the assay parameters on the mass spectrometer
<ol>
	<li>Use the application program (see the Table of Materials) to prepare a .xlsx file containing the predicted signal m/z value for importing. 
	NOTE: The settings in FILE I.xlsx (Table 2) are example settings for the UDG assay of G:U substrate in section 2.</li>
	<li>Use the application program to create and define a new UDG assay by right-clicking Import Assay Group in Designer Format option and selecting the .xlsx file from the dropdown list (e.g., FILE I.xlsx from step 4.1).</li>
	<li>Right-click the Customer:Project:Plate button and click on the top of the dropdown option tree to establish a new assay plate. In the dialog box, type in a file name (e.g., CTT20210620 for the lab code and assay date), and in the plate type dropdown, select the 384-well plate type and press OK. Look for a blank plate to appear on the right of the screen.</li>
	<li>Click the Assay option; select the assay (e.g., FILE I.xlsx) from the dropdown list.</li>
	<li>To assign the selected assay (e.g., FILE I.xlsx) to each sample spot position on the plate, move the cursor to each position of the blank plate, click to highlight the well, and right-click to select Add Plex.</li>
	<li>Use a desktop or laptop computer to prepare a working list in .xlsx format with no header (e.g., 0620.xlsx of Table 3) for all the samples on the chip from step 2.7. Click the Add New Sample Project button; select the file (e.g., 0620.xlsx) from the dropdown list to import the working list.</li>
	<li>Look for all the test sample codes in the working list (e.g., from 0620.xlsx) on the left of the screen. Click the sample code in the working list, and right-click on the corresponding position of the plate to link the tests to each position.</li>
</ol>
5. Mass spectrometer operation
<ol>
	<li>Use the application program to link the mass spectrometer (see the Table of Materials) to the sample chip (from section 3) to be analyzed.</li>
	<li>Click on the default setting. In the dialog box, type in a file name from step 4.3 (e.g., CTT20210620); in the Experiment Name, type in the chip ID in the Chip Barcode, and save the settings.</li>
	<li>Start the mass spectrometer control program (see the Table of Materials).</li>
	<li>Push the In/Out button of the mass spectrometer and let the deck extend. Take out the chip holder and insert the sample chip from step 3.4 into the chip holder. Place the loaded chip holder onto the extended deck and push the In/Out button for the sample chip to enter the instrument.</li>
	<li>Double-click the Acquire icon of the application program. In the Acquire window, click the auto run tab to start the instrument and acquire mass spectra from the samples on the chip.</li>
</ol>
6. Viewing mass spectra and analyzing the data
<ol>
	<li>Run the data analysis program (see the Table of Materials).</li>
	<li>Browse the database tree and select the chip ID from step 5.2. Click to highlight a target well on the chip, and click the spectrum icon to show the mass spectrum.</li>
	<li>Right-click to choose Customization Dialog to crop a specific range of spectrum in a new window. Click on the X-Axis to type in the upper and lower limits of m/z, and press OK to show the specified range spectrum, including the signals of interest. 
	NOTE: The amount of DNA is proportional to the peak intensity at each m/z value unit.</li>
	<li>Measure the peak height of signals&#39; m/z values corresponding to U substrate, AP-product, and template. Click on the peak and view the peak height in the upper left corner of the screen. 
	NOTE: A spectrum of 1,600 width/1,200 unit is a reasonable dimension for inspection on a computer screen as well as for recordkeeping.</li>
	<li>To save the spectrum for recordkeeping, right-click Export and select JPEG file type in the dropdown list. Click on Destination and Browse Disc to select the storage device in the dropdown list (e.g., flash disc E:). Type the file name (e.g., 0620_1-2.jpg) and click Export.</li>
	<li>If required, print out an exported JPEG file and measure the peak height manually using a ruler.</li>
</ol>

The authors have no conflicts of interest to disclose.

This paper provides a detailed procedure for using a UDG MALDI-TOF MS assay method to directly detect AP-containing DNA products. The main advantages of this method are that uracil-containing substrates are label-free, scalable, easy to work with, and afford greater flexibility in substrate design.
The UDG supplier-recommended phenol/chloroform extraction enables inactivation of the enzyme to prevent degradation of product DNA. However, the phenol extraction protocol involves tedious phase-sep...

Although uracil is a normal base in RNA, it is a common and highly mutagenic lesion in genomic DNA. Uracil can arise from spontaneous/enzymatic hydrolytic deamination of a deoxycytidine. In each living cell, this deamination occurs 100-500 times per day under physiological conditions1,2. If these alterations are not repaired, there can be a change in the DNA sequence composition, causing mutation. As uracil in DNA prefers to pair with dATP during replication, if cytosine deaminates to uracil, in two replication events, there will be a new G:C to A:T transition mutation in half of the progeny DNA3.
Among the cellular strategies to maintain genetic stability, base excision repair (BER) is an essential mechanism that repairs damaged bases, such as uracil, in DNA4. BER is a highly evolutionarily conserved process. There are two general BER pathways: the short-patch pathway leading to a repair tract of a single nucleotide and the long-patch pathway that produces a repair tract of at least two nucleotides5. BER is a coordinated mechanism that occurs in several steps. The first step in BER is the enzymatic hydrolysis of the damaged nucleotide base by a damage-specific DNA glycosylase to generate an apurinic/apyrimidinic (AP) intermediate site6. This is followed by the cleavage of the sugar-phosphate backbone at the AP site by an endonuclease, clean-up of the DNA ends by a lyase, gap-filling by a DNA polymerase, and sealing the final nick by a ligase5.
Uracil-DNA glycosylase (UDG) hydrolyzes the uracil from uracil-containing DNA for BER in Escherichia coli. Conventional UDG assays using radiolabeled DNA involving different separation techniques6,7,8,9,10,11,12,13 are usually time-consuming, labor-intensive, with costly labeling reagents, complicated procedures, and requiring intensive training and practice to reduce risks of exposure to radioactive materials. Fluorometric oligonucleotide assays have been developed as a replacement for radioisotope labeling14, in addition to molecular beacons and F&#246;rster resonance energy transfer technology15,16,17,18,19,20. However, specific labeling is required for all the aforementioned methods. Recently label-free biosensors assays21,22,23 and colorimetric methods based on the formation of a G-quadruplex24,25,26 have been developed. However, multiple A:U pairs or specially designed sequences in the probes complicate enzyme unit definition.
MALDI-TOF MS is a technology that could be of great use in DNA analysis. Applications developed include single-nucleotide polymorphism genotyping27,28, modified nucleotide analysis29, and DNA repair intermediate identification30,31,32,33,34. MALDI-TOF MS should be readily adopted for DNA glycosylase analysis to detect AP-site-containing DNA products. However, AP-sites in DNA are prone to strand break under many experimental conditions33. A UDG assay is presented here using MALDI-TOF MS to directly measure AP site production without significant strand-break noise. This label-free method is easy to work with and has a high potential for the pharmaceutical application of DNA glycosylase inhibitor screening.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>2-Amino-2-hydroxymethyl-propane-1,3-diol (Tris base)</td>
			<td>J.T baker</td>
			<td></td>
			<td>Protocol 1,2</td>
		</tr>
		<tr>
			<td>Autoclaved deionized water</td>
			<td>MILLIPORE</td>
			<td></td>
			<td>Protocol 1,2</td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>J.T Baker</td>
			<td></td>
			<td>Protocol 1,2</td>
		</tr>
		<tr>
			<td>Gloves</td>
			<td>AQUAGLOVE</td>
			<td></td>
			<td>Protocol 1,2,3</td>
		</tr>
		<tr>
			<td>Hydrochloric acid (HCl)</td>
			<td>SIGMA</td>
			<td></td>
			<td>Protocol 1,2</td>
		</tr>
		<tr>
			<td>Ice bucket</td>
			<td>Taiwan.Inc</td>
			<td></td>
			<td>Protocol 2</td>
		</tr>
		<tr>
			<td>Low retention pipette tips(0.5-10 &#181;L)</td>
			<td>extra gene</td>
			<td></td>
			<td>Protocol 1,2</td>
		</tr>
		<tr>
			<td>Low retention pipette tips(1,250 &#181;L)</td>
			<td>national scientific supply co, Inc.</td>
			<td></td>
			<td>Protocol 1,2</td>
		</tr>
		<tr>
			<td>Low retention pipette tips(200 &#181;L)</td>
			<td>national scientific supply co, Inc.</td>
			<td></td>
			<td>Protocol 1,2</td>
		</tr>
		<tr>
			<td>MassARRAY</td>
			<td>&#160;Agena Bioscience, CA</td>
			<td></td>
			<td>Protocol 4, 5 
			Mass spectrometry control programs include Typer Chip Linker, SpectroACQUIRE, and Start RT Process.</td>
		</tr>
		<tr>
			<td>MassARRAY Nanodispenser</td>
			<td>AAT Bioquest, Inc.</td>
			<td>RS1000</td>
			<td>Protocol 3</td>
		</tr>
		<tr>
			<td>Microcentrifuge</td>
			<td>Kubota</td>
			<td></td>
			<td>Protocol 2</td>
		</tr>
		<tr>
			<td>Microcentrifuge</td>
			<td>Clubio</td>
			<td></td>
			<td>Protocol 2</td>
		</tr>
		<tr>
			<td>Microcentrifuge tube (1.5 mL)</td>
			<td>National scientific supply co, Inc.</td>
			<td></td>
			<td>Protocol 2</td>
		</tr>
		<tr>
			<td>Microcentrifuge tube rack</td>
			<td>Taiwan.Inc</td>
			<td></td>
			<td>Protocol 1,2</td>
		</tr>
		<tr>
			<td>Micropipette&#160; (P1000)</td>
			<td>Gilson</td>
			<td></td>
			<td>Protocol 1,2</td>
		</tr>
		<tr>
			<td>Micropipette&#160; (P2)</td>
			<td>Gilson</td>
			<td></td>
			<td>Protocol 1,2</td>
		</tr>
		<tr>
			<td>Micropipette (P10)</td>
			<td>Gilson</td>
			<td></td>
			<td>Protocol 1,2</td>
		</tr>
		<tr>
			<td>Micropipette (P100)</td>
			<td>Gilson</td>
			<td></td>
			<td>Protocol 1,2</td>
		</tr>
		<tr>
			<td>Micropipette (P200)</td>
			<td>Gilson</td>
			<td></td>
			<td>Protocol 1,2</td>
		</tr>
		<tr>
			<td>Micropipette (SL2)</td>
			<td>Rainin</td>
			<td></td>
			<td>Protocol 1,2</td>
		</tr>
		<tr>
			<td>Oligonucleotides</td>
			<td>Integrated DNA Technologies (Singapore)</td>
			<td></td>
			<td>Protocol 1,2</td>
		</tr>
		<tr>
			<td>Quest Graph IC50 Calculator (v.1)</td>
			<td>AAT Bioquest, Inc.</td>
			<td></td>
			<td>Fig. 4 
			https://www.aatbio.com/tools/ic50-calculator-v1</td>
		</tr>
		<tr>
			<td>Sodium hydroxide (NaOH)</td>
			<td>WAKO</td>
			<td></td>
			<td>Protocol 2</td>
		</tr>
		<tr>
			<td>SpectroCHIP array</td>
			<td>&#160;Agena Bioscience, CA</td>
			<td>#01509</td>
			<td>Protocol 3, 5</td>
		</tr>
		<tr>
			<td>Timer</td>
			<td>Taiwan.Inc</td>
			<td></td>
			<td>Protocol 2</td>
		</tr>
		<tr>
			<td>Typer 4.0 software</td>
			<td>&#160;Agena Bioscience, CA</td>
			<td>#10145</td>
			<td>Protocol 6 
			Typer 4.0 consists four programs including Assay Designer, Assay Editor, Plate Editor, and Typer Analyzer.</td>
		</tr>
		<tr>
			<td>UDG Reaction Buffer (10x)</td>
			<td>New England Biolabs, MA</td>
			<td>B0280S</td>
			<td>Protocol 2</td>
		</tr>
		<tr>
			<td>Uracil Glycosylase Inhibitor</td>
			<td>New England Biolabs, MA</td>
			<td>M0281S</td>
			<td>Protocol 2</td>
		</tr>
		<tr>
			<td>Uracil-DNA Glycosylase</td>
			<td>New England Biolabs, MA</td>
			<td>M0280L</td>
			<td>Protocol 2</td>
		</tr>
		<tr>
			<td>UV-VISBLE spectrophotometer UV-1601</td>
			<td>SHIMADZU</td>
			<td></td>
			<td>Protocol 1</td>
		</tr>
		<tr>
			<td>Water bath</td>
			<td>ZETA ZC-4000 (Taiwan.Inc)</td>
			<td></td>
			<td>Protocol 2</td>
		</tr>
	</tbody>
</table>

uracil-dna glycosylase assay by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis

Uracil-DNA glycosylase (UDG) is a key component in the base excision repair pathway for the correction of uracil formed from hydrolytic deamination of cytosine. Thus, it is crucial for genome integrity maintenance. A highly specific, non-labeled, non-radio-isotopic method was developed to measure UDG activity. A synthetic DNA duplex containing a site-specific uracil was cleaved by UDG and then subjected to Matrix-assisted Laser Desorption/Ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis. A protocol was established to preserve the apurinic/apyrimidinic site (AP) product in DNA without strand break. The change in the m/z value from the substrate to the product was used to evaluate uracil hydrolysis by UDG. A G:U substrate was used for UDG kinetic analysis yielding the Km = 50 nM, Vmax = 0.98 nM/s, and Kcat = 9.31 s-1. Application of this method to a uracil glycosylase inhibitor (UGI) assay yielded an IC50 value of 7.6 pM. The UDG specificity using uracil at various positions within single-stranded and double-stranded DNA substrates demonstrated different cleavage efficiencies. Thus, this simple, rapid, and versatile MALDI-TOF MS method could be an excellent reference method for various monofunctional DNA glycosylases. It also has the potential as a tool for DNA glycosylase inhibitor screening.

Templates and substrates 
Taking synthetic oligonucleotides with U in the center (U+9) paired with a G template as an example (Figure 1A), a blank control of equimolar amounts of template and uracil-containing substrate can be used for quality control of synthetic oligonucleotide purity (Figure&#160;1B; the signals match the designated m/z and the low background noise). For the MS data analysis, the peak heights were measured (<strong class...

Watch this Scientific Journal Video about Uracil-DNA Glycosylase Assay by Matrix-assisted Laser Desorption/Ionization Time-of-flight Mass Spectrometry Analysis at JoVE.com

Uracil-DNA Glycosylase Assay by Matrix-assisted Laser Desorption/Ionization Time-of-flight Mass Spectrometry Analysis

A non-labeled, non-radio-isotopic method to assay uracil-DNA glycosylase activity was developed using MALDI-TOF mass spectrometry for direct apurinic/apyrimidinic site-containing product analysis. The assay proved to be quite simple, specific, rapid, and easy to use for DNA glycosylase measurement.

Uracil-DNA glycosylase (UDG) is a key component in the base excision repair pathway for the correction of uracil formed from hydrolytic deamination of ...

uracil-dna-glycosylase-assay-matrix-assisted-laser

Research

JoVE Journal

Biochemistry

3.7K Views.  National Taiwan University. A non-labeled, non-radio-isotopic method to assay uracil-DNA glycosylase activity was developed using MALDI-TOF mass spectrometry for direct apurinic/apyrimidinic site-containing product analysis. The assay proved to be quite simple, specific, rapid, and easy to use for DNA glycosylase measurement. 

Video: Uracil-DNA Glycosylase Assay by Matrix-assisted Laser Desorption/Ionization Time-of-flight Mass Spectrometry Analysis

Time-resolved ElectroSpray Ionization Hydrogen-deuterium Exchange Mass Spectrometry for Studying Protein Structure and Dynamics

Intrinsically disordered proteins (IDPs) have long been a challenge to structural biologists due to their lack of stable secondary structure elements. Hydrogen-Deuterium Exchange (HDX) measured at rapid time scales is uniquely suited to detect structures and hydrogen bonding networks that are briefly populated, allowing for the characterization of transient conformers in native ensembles. Coupling of HDX to mass spectrometry offers several key advantages, including high sensitivity, low sample consumption and no restriction on protein size. This technique has advanced greatly in the last several decades, including the ability to monitor HDX labeling times on the millisecond time scale. In addition, by incorporating the HDX workflow onto a microfluidic platform housing an acidic protease microreactor, we are able to localize dynamic properties at the peptide level. In this study, Time-Resolved ElectroSpray Ionization Mass Spectrometry (TRESI-MS) coupled to HDX was used to provide a detailed picture of residual structure in the tau protein, as well as the conformational shifts induced upon hyperphosphorylation.

Conformational flexibility plays a critical role in protein function. Herein, we describe the use of time-resolved electrospray ionization mass spectrometry coupled to hydrogen-deuterium exchange for probing the rapid structural changes that drive function in ordered and disordered proteins.

Intrinsically disordered proteins (IDPs) have long been a challenge to structural biologists due to their lack of stable secondary structure elements. ...

Resolving Affinity Purified Protein Complexes by Blue Native PAGE and Protein Correlation Profiling

Most proteins act in association with others; hence, it is crucial to characterize these functional units in order to fully understand biological processes. Affinity purification coupled to mass spectrometry (AP-MS) has become the method of choice for identifying protein-protein interactions. However, conventional AP-MS studies provide information on protein interactions, but the organizational information is lost. To address this issue, we developed a strategy to unravel the distinct functional assemblies a protein might be involved in, by resolving affinity-purified protein complexes prior to their characterization by mass spectrometry. Protein complexes isolated through affinity purification of a bait protein using an epitope tag and competitive elution are separated through blue native electrophoresis. Comparison of protein migration profiles through correlation profiling using quantitative mass spectrometry allows assignment of interacting proteins to distinct molecular entities. This method is able to resolve protein complexes of close molecular weights that might not be resolved by traditional chromatographic techniques such as gel filtration. With little more work than conventional AP-geLC-MS/MS, we demonstrate this strategy may in many cases be adequate for obtaining protein complex topological information concomitantly to identifying protein interactions.

Here we present protocols for affinity purification of protein complexes and their separation by blue native PAGE, followed by protein correlation profiling using label free quantitative mass spectrometry. This method is useful to resolve interactomes into distinct protein complexes.

Most proteins act in association with others; hence, it is crucial to characterize these functional units in order to fully understand biological ...

A Tandem Liquid Chromatography&#8211;Mass Spectrometry-based Approach for Metabolite Analysis of Staphylococcus aureus

In an effort to thwart bacterial pathogens, hosts often limit the availability of nutrients at the site of infection. This limitation can alter the abundances of key metabolites to which regulatory factors respond, adjusting cellular metabolism. In recent years, a number of proteins and RNA have emerged as important regulators of virulence gene expression. For example, the CodY protein responds to levels of branched-chain amino acids and GTP and is widely conserved in low G+C Gram-positive bacteria. As a global regulator in Staphylococcus aureus, CodY controls the expression of dozens of virulence and metabolic genes. We hypothesize that S. aureus uses CodY, in part, to alter its metabolic state in an effort to adapt to nutrient-limiting conditions potentially encountered in the host environment. This manuscript describes a method for extracting and analyzing metabolites from S. aureus using liquid chromatography coupled with mass spectrometry, a protocol that was developed to test this hypothesis. The method also highlights best practices that will ensure rigor and reproducibility, such as maintaining biological steady state and constant aeration without the use of continuous chemostat cultures. Relative to the USA200 methicillin-susceptible S. aureus isolate UAMS-1 parental strain, the isogenic codY mutant exhibited significant increases in amino acids derived from aspartate (e.g., threonine and isoleucine) and decreases in their precursors (e.g., aspartate and O-acetylhomoserine). These findings correlate well with transcriptional data obtained with RNA-seq analysis: genes in these pathways were up-regulated between 10- and 800-fold in the codY null mutant. Coupling global analyses of the transcriptome and the metabolome can reveal how bacteria alter their metabolism when faced with environmental or nutritional stress, providing potential insight into the physiological changes associated with nutrient depletion experienced during infection. Such discoveries may pave the way for the development of novel anti-infectives and therapeutics.

A Tandem Liquid Chromatography&amp;#8211;Mass Spectrometry-based Approach for Metabolite Analysis of &lt;em&gt;Staphylococcus aureus&lt;/em&gt;

Here we describe a protocol for the extraction of metabolites from Staphylococcus aureus and their subsequent analysis via liquid chromatography and mass spectrometry.

In an effort to thwart bacterial pathogens, hosts often limit the availability of nutrients at the site of infection. This limitation can alter the ...

Lipid Droplet Isolation for Quantitative Mass Spectrometry Analysis

Lipid droplets are vital to the replication of a variety of different pathogens, most prominently the Hepatitis C Virus (HCV), as the putative site of virion morphogenesis. Quantitative lipid droplet proteome analysis can be used to identify proteins that localize to or are displaced from lipid droplets under conditions such as virus infections. Here, we describe a protocol that has been successfully used to characterize the changes in the lipid droplet proteome following infection with HCV. We use Stable Isotope Labeling with Amino Acids in Cell Culture (SILAC) and thus label the complete proteome of one population of cells with &#34;heavy&#34; amino acids to quantitate the proteins by mass spectrometry. For lipid droplet isolation, the two cell populations (i.e.&#160;HCV-infected/&#34;light&#34; amino acids and uninfected control/&#34;heavy&#34; amino acids) are mixed 1:1 and lysed mechanically in hypotonic buffer. After removing the nuclei and cell debris by low speed centrifugation, lipid droplet-associated proteins are enriched by two subsequent ultracentrifugation steps followed by three washing steps in isotonic buffer. The purity of the lipid droplet fractions is analyzed by western blotting with antibodies recognizing different subcellular compartments. Lipid droplet-associated proteins are then separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) followed by Coomassie staining. After tryptic digest, the peptides are quantified by liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS). Using this method, we identified proteins recruited to lipid droplets upon HCV infection that might represent pro- or antiviral host factors. Our method can be applied to a variety of different cells and culture conditions, such as infection with pathogens, environmental stress, or drug treatment.

Lipid droplets are important organelles for the replication of several pathogens, including the Hepatitis C Virus (HCV). We describe a method to isolate lipid droplets for quantitative mass spectrometry of associated proteins; it can be used under a variety of conditions, such as virus infection, environmental stress, or drug treatment.

Lipid droplets are vital to the replication of a variety of different pathogens, most prominently the Hepatitis C Virus (HCV), as the putative site of ...

Glycoproteomics of the Extracellular Matrix: A Method for Intact Glycopeptide Analysis Using Mass Spectrometry

Fibrosis is a hallmark of many cardiovascular diseases and is associated with the exacerbated secretion and deposition of the extracellular matrix (ECM). Using proteomics, we have previously identified more than 150 ECM and ECM-associated proteins in cardiovascular tissues. Notably, many ECM proteins are glycosylated. This post-translational modification affects protein folding, solubility, binding, and degradation. We have developed a sequential extraction and enrichment method for ECM proteins that is compatible with the subsequent liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis of intact glycopeptides. The strategy is based on sequential incubations with NaCl, SDS for tissue decellularization, and guanidine hydrochloride for the solubilization of ECM proteins. Recent advances in LC-MS/MS include fragmentation methods, such as combinations of higher-energy collision dissociation (HCD) and electron transfer dissociation (ETD), which allow for the direct compositional analysis of glycopeptides of ECM proteins. In the present paper, we describe a method to prepare the ECM from tissue samples. The method not only allows for protein profiling but also the assessment and characterization of glycosylation by MS analysis.

This paper describes a methodology to prepare cardiovascular tissue samples for MS analysis that allows for (1) the analysis of ECM protein composition, (2) the identification of glycosylation sites, and (3) the compositional characterization of glycan forms. This methodology can be applied, with minor modifications, to the study of the ECM in other tissues.

Fibrosis is a hallmark of many cardiovascular diseases and is associated with the exacerbated secretion and deposition of the extracellular matrix (ECM). ...

Optimal Preparation of Formalin Fixed Samples for Peptide Based Matrix Assisted Laser Desorption/Ionization Mass Spectrometry Imaging Workflows

The use of matrix-assisted laser desorption/ionization, mass spectrometry imaging (MALDI MSI) has rapidly expanded, since this technique analyzes a host of biomolecules from drugs and lipids to N-glycans. Although various sample preparation techniques exist, detecting peptides from formaldehyde preserved tissues remains one of the most difficult challenges for this type of mass spectrometric analysis. For this reason, we have created and optimized a robust methodology that preserves the spatial information contained within the sample, while eliciting the greatest number of ionizable peptides. We have also aimed to achieve this in a cost effective and simple way, thereby eliminating potential bias or preparation error, which can occur when using automated instrumentation. The end result is a reproducible and inexpensive protocol.

This protocol describes a reproducible and reliable method for the sublimation-based preparation of formalin fixed tissue destined for imaging mass spectrometry.

The use of matrix-assisted laser desorption/ionization, mass spectrometry imaging (MALDI MSI) has rapidly expanded, since this technique analyzes a host ...

Proofreading and DNA Repair Assay Using Single Nucleotide Extension and MALDI-TOF Mass Spectrometry Analysis

The maintenance of the genome and its faithful replication is paramount for conserving genetic information. To assess high fidelity replication, we have developed a simple non-labeled and non-radio-isotopic method using a matrix-assisted laser desorption ionization with time-of-flight (MALDI-TOF) mass spectrometry (MS) analysis for a proofreading study. Here, a DNA polymerase [e.g., the Klenow fragment (KF) of Escherichia coli DNA polymerase I (pol I) in this study] in the presence of all four dideoxyribonucleotide triphosphates is used to process a mismatched primer-template duplex. The mismatched primer is then proofread/extended and subjected to MALDI-TOF MS. The products are distinguished by the mass change of the primer down to single nucleotide variations. Importantly, a proofreading can also be determined for internal single mismatches, albeit at different efficiencies. Mismatches located at 2-4-nucleotides (nt) from the 3' end were efficiently proofread by pol I, and a mismatch at 5 nt from the primer terminus showed only a partial correction. No proofreading occurred for internal mismatches located at 6 - 9 nt from the primer 3' end. This method can also be applied to DNA repair assays (e.g., assessing a base-lesion repair of substrates for the endo V repair pathway). Primers containing 3' penultimate deoxyinosine (dI) lesions could be corrected by pol I. Indeed, penultimate T-I, G-I, and A-I substrates had their last 2 dI-containing nucleotides excised by pol I before adding a correct ddN 5'-monophosphate (ddNMP) while penultimate C-I mismatches were tolerated by pol I, allowing the primer to be extended without repair, demonstrating the sensitivity and resolution of the MS assay to measure DNA repair.

A non-labeled, non-radio-isotopic method for DNA polymerase proofreading and a DNA repair assay was developed by using high-resolution MALDI-TOF mass spectrometry and a single nucleotide extension strategy. The assay proved to be very specific, simple, rapid, and easy to perform for proofreading and repair patches shorter than 9-nucleotides.

The maintenance of the genome and its faithful replication is paramount for conserving genetic information. To assess high fidelity replication, we have ...

Detection of Protein Ubiquitination Sites by Peptide Enrichment and Mass Spectrometry

The posttranslational modification of proteins by the small protein ubiquitin is involved in many cellular events. After tryptic digestion of ubiquitinated proteins, peptides with a diglycine remnant conjugated to the epsilon amino group of lysine (&#39;K-&#949;-diglycine&#39; or simply &#39;diGly&#39;) can be used to track back the original modification site. Efficient immunopurification of diGly peptides combined with sensitive detection by mass spectrometry has resulted in a huge increase in the number of ubiquitination sites identified up to date. We have made several improvements to this workflow, including offline high pH reverse-phase fractionation of peptides prior to the enrichment procedure, and the inclusion of more advanced peptide fragmentation settings in the ion routing multipole. Also, more efficient cleanup of the sample using a filter-based plug in order to retain the antibody beads results in a greater specificity for diGly peptides. These improvements result in the routine detection of more than 23,000 diGly peptides from human cervical cancer cells (HeLa) cell lysates upon proteasome inhibition in the cell. We show the efficacy of this strategy for in-depth analysis of the ubiquitinome profiles of several different cell types and of in vivo samples, such as brain tissue. This study presents an original addition to the toolbox for protein ubiquitination analysis to uncover the deep cellular ubiquitinome.

We present a method for the purification, detection, and identification of diGly peptides that originate from ubiquitinated proteins from complex biological samples. The presented method is reproducible, robust, and outperforms published methods with respect to the level of depth of the ubiquitinome analysis.

The posttranslational modification of proteins by the small protein ubiquitin is involved in many cellular events. After tryptic digestion of ...

Nitropeptide Profiling and Identification Illustrated by Angiotensin II

Protein nitration is one of the most important post-translational modifications (PTM) on tyrosine residues and it can be induced by chemical actions of reactive oxygen species (ROS) and reactive nitrogen species (RNS) in eukaryotic cells. Precise identification of nitration sites on proteins is crucial for understanding the physiological and pathological processes related to protein nitration, such as inflammation, aging, and cancer. Since the nitrated proteins are of low abundance in cells even under induced conditions, no universal and efficient methods have been developed for the profiling and identification of protein nitration sites. Here we describe a protocol for nitropeptide enrichment by using a chemical reduction reaction and biotin labeling, followed by high resolution mass spectrometry. In our method, nitropeptide derivatives can be identified with high accuracy. Our method exhibits two advantages compared to the previously reported methods. First, dimethyl labeling is used to block the primary amine on nitropeptides, which can be used to generate quantitative results. Second, a disulfide bond containing NHS-biotin reagent is used for the enrichment, which can be further reduced and alkylated to enhance the detection signal on a mass spectrometer. This protocol has been successfully applied to the model peptide Angiotensin II in the current paper.

Proteomic profiling of tyrosine-nitrated proteins has been a challenging technique due to the low abundance of the 3-nitrotyrosine modification. Here we describe a novel approach for nitropeptide enrichment and profiling by using Angiotensin II as the model. This method can be extended for other in vitro or in vivo systems.

Protein nitration is one of the most important post-translational modifications (PTM) on tyrosine residues and it can be induced by chemical actions of ...

Quantitative Analysis of the Cellular Lipidome of Saccharomyces Cerevisiae Using Liquid Chromatography Coupled with Tandem Mass Spectrometry

Lipids are structurally diverse amphipathic molecules that are insoluble in water. Lipids are essential contributors to the organization and function of biological membranes, energy storage and production, cellular signaling, vesicular transport of proteins, organelle biogenesis, and regulated cell death. Because the budding yeast Saccharomyces cerevisiae is a unicellular eukaryote amenable to thorough molecular analyses, its use as a model organism helped uncover mechanisms linking lipid metabolism and intracellular transport to complex biological processes within eukaryotic cells. The availability of a versatile analytical method for the robust, sensitive, and accurate quantitative assessment of major classes of lipids within a yeast cell is crucial for getting deep insights into these mechanisms. Here we present a protocol to use liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) for the quantitative analysis of major cellular lipids of S. cerevisiae. The LC-MS/MS method described is versatile and robust. It enables the identification and quantification of numerous species (including different isobaric or isomeric forms) within each of the 10 lipid classes. This method is sensitive and allows identification and quantitation of some lipid species at concentrations as low as 0.2 pmol/&#181;L. The method has been successfully applied to assessing lipidomes of whole yeast cells and their purified organelles. The use of alternative mobile phase additives for electrospray ionization mass spectrometry in this method can increase the efficiency of ionization for some lipid species and can be therefore used to improve their identification and quantitation.

Quantitative Analysis of the Cellular Lipidome of Saccharomyces Cerevisiae Using Liquid Chromatography Coupled with Tandem Mass Spectrometry

We present a protocol using liquid chromatography coupled with tandem mass spectrometry to identify and quantify major cellular lipids in Saccharomyces cerevisiae. The described method for a quantitative assessment of major lipid classes within a yeast cell is versatile, robust, and sensitive.

Lipids are structurally diverse amphipathic molecules that are insoluble in water. Lipids are essential contributors to the organization and function of ...